The long term goal of this project is to develop new technology that will allow rapid user- defined patterning of proteins on cell culture substrates at dimensions ranging from 50 nanometers to millimeters. Patterned cell culture substrates are emerging as a powerful biomedical research tool. For example, they can be used to study how eukaryotic cells interact with molecules in the extracellular environment. However, the technology for making patterned substrates is still limited to a relatively small number of laboratories, and it is not available to the larger cell biology research community. Intelligent Substrates, Inc. has patent-pending rights to a patterning technology that is based on local laser inactivation of protein function. This technology is highly flexible, and will become part of a service that allows a user with no special training to order custom designed patterned substrates that will be delivered in 1-2 days. The technology has been demonstrated to work at dimensions of a few micrometers. However, the relevant length scale for the cell biological applications is ~10 nanometers and greater. Here we propose to advance ISI's laser-based technology to produce patterns with features with dimensions as small as 50 nm. The approach to this problem is first to build a UV excimer laser patterning system, and to establish the dose and laser intensity for optimal inactivation of two extracellular matrix proteins. Because there is an intensity threshold for inactivation, features of <100 nanometers can be made by effectively whittling the boundaries of the patterns of active matrix proteins. These substrates will be characterized using immunofluorescence microscopy and atomic force microscopy. The functionality of the patterns will be tested by culturing several different cell lines on them and by evaluating cell spreading and focal adhesion formation. Success of this research will enable ISI to offer a custom patterning service that permits scientists to order patterned tissue culture substrates with features as small as <100 nanometers. This will enable important biomedical research in a wide range of areas. Cell culture methods and technology, pioneered in the first half of the 20th century, have enabled the significant advances in public health, such as the discovery of the polio vaccine, and are the work horse of modern biomedical sciences. The research proposed here describes an enabling laser-based technology that allows scientists to control cell function creating patterns of active proteins with features the size of 10s of nanometers. Intelligent Substrates, Inc. plans to commercialize this method and produce custom patterns on demand for the biomedical research community.